The field of the present invention is fermentation systems. More specifically, the present invention relates to an apparatus and method for simultaneously fermenting multiple samples as part of a multiple process system.
Fermentation is a key technology in many fields and industries and is performed both on a mass production scale and on an experimental, bench top scale. For example, fermentation systems are used for the production of a large number of products such as antibiotics, vaccines, synthetic biopolymers, synthetic amino acids, and proteins. Fermentation technology is integral in the production of recombinant proteins using biological organisms such as E. coli and many other cell cultures. For example, production of commercial pharmaceuticals such as recombinant insulin (Eli Lilly), erythropoietin (Amgen), and interferon (Roche) all involve fermentation as an essential step.
Fermentation may be conducted on a production scale in order to make commercial quantities of pharmaceuticals or other products. Production scale processes emphasize limited human intervention and automation to increase output and efficiency. In an assembly line fashion, automated equipment enables high throughput processing of production scale amounts of material without disrupting the assembling, testing, or synthesizing process at each individual processing step. For example, automated liquid dispensers, aspirators, and specimen plate handlers facilitate the handling and testing of hundreds of thousands of samples per day, with limited human interaction with the actual sample from start to finish of the entire analysis process. In a further example, sample materials are automatically dispensed into multiple well specimen plates, reagents are added and removed via automated liquid dispensers and aspirators, and the specimen plates are transferred to each successive processing station by automated plate handlers. This increased production efficiency is premised in part on the viability of conducting the entire production process in the specimen plate. Similarly, automated procedures enable the synthesis of commercial pharmaceuticals from starting reagents to finished product without disrupting the production process with cumbersome, inefficient steps such as changing a sample vessel or transferring the sample materials manually to new sample vessels.
Rapid advances in biotechnology have enabled the development of high throughput alternatives to traditional laboratory bench top processes. Unfortunately, fermentation methods have not been amenable to automation because limits in current fermentation technology prevent the uninterrupted processing flow that characterizes automated high throughput systems. Existing fermentation systems typically involve multiple handling steps by either a batch processing method or a continuous processing method.
Current production scale batch processes involve first fermenting in large scale, bulk fermentation vessels, then processing the fermentation medium to isolate the desired fermentation product, followed by transferring this product into the production stream for further processing, and finally cleaning the fermentation apparatus for the next batch. In a large scale batch culture, it is generally necessary to provide a high initial concentration of nutrients in order to sustain cell growth over an extended time. As a result, substrate inhibition may occur in the early stages of cell growth and then may be followed by a nutrient deficiency in the late stages of fermentation. These disadvantages result in sub-optimal cell growth rates and fermentation yields. Another disadvantage of this method lies in the need to individually dispense the fermentation products from the bulk fermentation apparatus into separate sample vessels for further processing. Thus, by producing the fermentation product on a bulk scale, the fermentation product is not immediately available for automated processing. Further disadvantages include the decreased efficiency of both transferring the material to another sample vessel, as well as cleaning and sterilizing the fermentation apparatus for the next batch. These disadvantages result in increased production costs, inefficient production times and decreased yields.
Continuous batch processes involve siphoning off the fermentation product from the bulk fermentation vessel and continuously adding nutrients to the fermentation medium according to a calculated exponential growth curve. This curve, however, is merely an approximation that does not accurately predict cell growth in large, industrial scale quantities of fermentation medium. Consequently, due to the unpredictable nature of large scale fermentation environments, experienced personnel are required to monitor the feeding rate very closely. Changes in the fermentation environment may result in either poisoned fermentation products being siphoned off into the production stream or sub-optimal production yields due to starved fermentation mediums. As a further disadvantage, unpredictable fermentation product yields affect the accuracy of subsequent processing steps. For example, when the fermentation yield decreases, the amount of aspirating, the amount of reagent dispensed, or the centrifuge time is no longer optimized, or even predictable. Frequent or continuous monitoring of the fermentation process and adjustment of the fermentation conditions is often not practicable or efficient in a production scale process.
Fermentation remains a key processing step in a number of industries, particularly in biotechnology industries, and thus a need exists for incorporating fermentation processes into current multiple process systems, such as automated high throughput systems. A process that produces a precise, known, and repeatable amount of unpoisoned fermentation product with limited human interaction or sample vessel transfer is essential to integrating fermentation into modem production processes.
The present invention greatly alleviates the disadvantages of known fermentation systems by providing a fermentation apparatus that may be incorporated into high throughput processing systems.
Briefly, the fermentation apparatus is constructed to produce a known and repeatable amount of unpoisoned fermentation product using multiple fermentation vessels. To facilitate further processing and to be compatible with other product processing steps, the fermentation apparatus preferably has an array of sample vessels arranged in a container frame. The container frame may be configured to hold the sample vessels during fermentation and to transport the vessel array to or from another processing station.
The sample vessels may be arranged in the container frame in an array format to facilitate tracking of the sample vessels during the production process and to make the format of sample vessels compatible with other processing steps. In a preferred embodiment, a total of 96 sample vessels are arranged in an 8xc3x9712 format. An arrayed 96-member sample format is compatible with other methods for sample handling such as a 96-well microtiter plate. An 8xc3x9712 arrayed format is similarly compatible with sample handling formats designed for greater numbers of sample vessels, such as 384- and 1536-member sample formats, which are multiples of the 96-member sample format.
In a preferred embodiment a cannula array, having a number of cannula corresponding to the number of sample vessels in the sample vessel array, is configured such that each cannula may be placed inside a sample vessel. The cannula array may be attached to a gas distributor that delivers gases from a gas source through the cannula into the sample vessel. Depending on the gas delivered, either aerobic fermentation with agitation or oxygenation or anaerobic fermentation, i.e., with a nitrogen bubbler, for example, can be performed with the present invention. Because the fermentation volume for each individual sample vessel is smaller than a bulk fermentation apparatus, the fermentation product yields are more predictable and cell growth rates are more effectively optimized.
In one aspect the present invention features a method of fermenting a plurality of samples. The method involves the steps of processing a plurality of samples contained in associated sample vessels and fermenting the plurality of samples in the sample vessels. The processing can be done before and/or after the fermenting and the steps preferably are performed at different locations. Each sample preferably is a relatively small, non-bulk volume of material.
An important aspect of the present invention is that a plurality of samples being fermented in associated sample vessels will have similar yields and growth rates. Thus, the plurality of samples may be monitored and harvested at approximately the same, which minimizes the need for human intervention and which produces more predictable fermentation results.
In a preferred embodiment, the method involves the steps of: (a) providing a plurality of sample vessels, each sample vessel having a gripping surface and holding an associated fermentation sample; (b) transporting the plurality of sample vessels to a fermentation apparatus; (c) fermenting each fermentation sample by delivering oxygen, nitrogen or another gas capable of fermenting the sample from a gas source into each sample vessel; (d) manipulating each gripping surface located on each sample vessel; and (e) transferring the sample vessels from the fermentation apparatus to a processing station, wherein processing occurs within the sample vessels. The method may also include the steps of: (1) arranging the sample vessels into an array; (2) arranging a plurality of cannula into a corresponding array such that each cannula may be inserted into an associated corresponding sample vessel; (3) coupling the cannula array to a gas arrangement; and (4) positioning the gas arrangement such that each cannula is inserted into its associated corresponding sample vessel.
In another aspect, the invention provides a multiple process, multiple sample fermentation apparatus. The apparatus includes a means for processesing a plurality of samples contained in associated sample vessels and a means for fermenting the plurality of samples in the sample vessels. The apparatus preferably also includes a process controller.
In a preferred embodiment, the apparatus includes: (a) a fermentation processing station, constructed to receive an array of sample vessels; (b) an array of sample vessels, each sample vessel containing a sample, wherein each sample vessel is capable of undergoing multiple process steps before, during or after fermentation; (c) a gas arrangement positioned to provide oxygen, nitrogen or any other gas capable of fermenting the sample to each sample in the array of sample vessels; (d) a cannula array, configured such that each cannula is attached to the gas arrangement and positionable inside a sample vessel; and (e) a gripping surface on the sample vessel such that a transporter using the gripping surface can transfer the sample vessel from the fermentation processing station to another processing station, wherein the sample is processed directly from the sample vessel. The apparatus may also include: (1) a dispenser, positioned such that feed solution is dispensed within each sample vessel; (2) a sensor with a sample within the sample vessel; and (3) a process controller, configured such that the sensor and the dispenser are in communication with the controller.
Other aspects of the invention feature: (a) methods of moving an array of vessels to a fermentor, and then from a fermentor to a processing station with the assistance of a sample carrier; (b) methods of robotically moving an array of vessels to a fermentor, and then from a fermentor to a processing station; (c) methods of robotically moving a same array of sample vessels from a fermentor to a centrifuge where samples are centrifuged in the same sample vessels; (d) and a fermentor head apparatus.
In preferred embodiments, the fermentations are in sync in the sense that they are roughly growing at the same rate. This allows them to get harvested at the same time. This may be achieved, for example, by use of a negatively regulated promoter. Also, in another preferred embodiment, a certain media plus bubbler increases the amount of soluble protein produced. In yet another preferred embodiment, as noted above, a total of 96 sample vessels are arranged in an 8xc3x9712 format or a 384- or 1536-member sample formats is used.
One advantage of the present invention is that the sample vessels are capable of undergoing multiple process steps before, during or after fermentation. Each of these sample vessels has a gripping surface that a transporter uses to transfer the sample vessel to another processing station. These sample vessels are constructed such that post- and pre-fermentation steps may be conducted directly on the sample in the sample vessel. The compatibility of the sample vessel with other processing steps in the production eliminates increased production costs incurred both from first transferring fermentation product from a bulk fermentation vessel to a sample processing vessel, and then cleaning and sterilizing the bulk fermentation vessel. Further, eliminating a transfer step increases the efficiency of the overall process because of the decreased production time in not having to perform an extra transfer step and the increased yield from not losing any fermentation product in a transfer step.
Another advantage is that the fermentation apparatus may also be used in non-production scale environments where uninterrupted process flows are desirable. For example, the fermentation apparatus may be adapted to bench top processes on an experimental scale. This provides a further advantage of easily modifying the process later to an industrial scale by eliminating the step of redesigning the fermentation conditions that is usually required when scaling up a bench top process to a production scale process. Because the present invention utilizes smaller scale fermentation volumes, the unpredictability and unmanageability of bulk fermentation volumes is eliminated while still providing production scale quantities of fermentation product. A fermentation method or apparatus made according to the present invention may be utilized in any production, analysis, or system requiring multiple process steps.
Disadvantages resulting from increased production costs incurred from transferring fermentation product from a bulk fermentation vessel to a processing sample vessel are thus eliminated, as are the costs of cleaning and sterilizing a bulk fermentation apparatus for the next batch. According to the present invention, only the sample vessels will be cleaned at the end of the production process. In addition, valuable time is saved and yields are increased by not having to transfer a bulk fermentation product to a sample vessel that would be amenable to high throughput processing.
A further advantage is that calculation of exponential growth curves is more precise and reliable. This advantage is created because the fermentation volumes of the sample vessels are smaller than current production scale bulk fermentation systems. As a result, the nutrient feed may be more accurately optimized, resulting in the production of known and repeatable yields of fermentation product. In addition, each sample vessel may be equipped with sensors that transmit data to a controller, enabling the apparatus to respond to suboptimal fermentation conditions by appropriately adjusting environmental parameters. The present invention uses relatively small volumes by fermenting in a sample vessel and thereby eliminates the erratic fluctuations in environmental conditions that lead to unpredictability of fermentation growth yields. As a result, for example, the amount of aspirating, the amount of reagent dispensed, or the centrifuge time may now be predicted and optimized, leading to a more efficient and reliable system. The steps of monitoring of the fermentation process to determine the fermentation yield and monitoring or adjusting further processes downstream, such as dispensing or aspirating steps based on the amount of fermentation product, are eliminated when using smaller volume fermentation batches.
Another added advantage stems from the size of the fermentation batches. Because these fermentation batches are relatively small compared to the bulk fermentation vessels currently being used, known amounts of nutrients may be calculated to optimize the fermentation yield and known fermentation yields may be calculated on a predictable and repeatable basis. This reliability in calculating a fermentation yield enables the optimization of centrifuge times, dispensing accurate amounts of reagent, and aspirating accurate amounts of liquid that is otherwise not possible in current bulk fermentation systems. Without a reliable and repeatable fermentation product yield, it is very difficult to engineer a high throughput system involving many processing steps where each processing step, such as the amount dispensed or the time centrifuged, would otherwise vary according to a fluctuating fermentation yield. The present invention overcomes these difficulties by providing predictable and repeatable fermentation yields upon which to calculate and optimize subsequent processing steps, such as those used in a high throughput system.
These and other features and advantages of the present invention will be appreciated from review of the following detailed description of the invention, along with the accompanying figures in which like reference numerals refer to like parts throughout.